US 2013/0155403 A1 · Integrated Nucleic Acid Analysis

Abstract

The present disclosure provides fully integrated microfluidic systems to perform nucleic acid analysis. These processes include sample collection, nucleic acid extraction and purification, amplification, sequencing, and separation and detection. The present disclosure also provides optical detection systems and methods for separation and detection of biological molecules. In particular, the various aspects of the invention enable the simultaneous separation and detection of a plurality of biological molecules, typically fluorescent dye-labeled nucleic acids, within one or a plurality of microfluidic chambers or channels. The nucleic acids can be labeled with at least 6 dyes, each having a unique peak emission wavelength. The present systems and methods are particularly useful for DNA fragment sizing applications such as human identification by genetic fingerprinting and DNA sequencing applications such as clinical diagnostics.

Claims45

Dependent claims

1-46. (canceled)

A system to simultaneously detect fragments from sequencing of at least two DNA templates in a single separation channel, comprising:

(i) an integrated biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 8 or more fluorescent dyes are used to generate two sets of nucleic acid fragments labeled with said 8 or more fluorescent dyes, said dyes being members of at least two 4-dye containing sets, such that the emission wavelengths of the dyes in the at least two sets are different and said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position;
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate;
and
(iv) a wavelength dispersive element positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to at least eight detectors positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

2 more elements

A system to detect components separated by electrophoresis comprising:

(i) a biochip comprising a thermal cycling chamber adapted for multiplexed amplification such that primers labeled with 6 or more fluorescent dyes are used to generate amplified nucleic acid fragments labeled with said 6 or more fluorescent dyes, said thermal cycling chamber in fluid communication with at least one channel on said biochip, wherein each channel comprises a detection position, and a second chamber adapted for additional processing, said processing selected from the group consisting of:
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) post-nucleic acid amplification cleanup;
(v) pre-nucleic acid sequencing cleanup;
(vi) nucleic acid sequencing;
(vii) post-nucleic acid sequencing cleanup;
(viii) reverse transcription;
(ix) pre-reverse transcription cleanup;
(x) post-reverse transcription cleanup;
(xi) nucleic acid ligation;
(xii) nucleic acid hybridization;
and
(xiii) quantification;
and
(ii) one or more light sources positioned for illuminating one or a plurality of detection positions over a physical space on said biochip;
(iii) one or a plurality of first optical elements positioned over said physical space for collecting and directing light emanating from the one or plurality of detection positions on said substrate;
and
(iv) a wavelength dispersive element positioned to disperse the wavelength components of the fluorescence over a physical space according to light wavelength, and to provide at least a portion of the dispersed wavelength components to at least six detectors positioned to detect fluorescence from said labeled amplified nucleic acid fragments.

17 more elements

An integrated biochip system comprising:

(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein
the first reaction chamber is adapted for
(xiv) nucleic acid extraction;
(xv) nucleic acid purification;
(xvi) pre-nucleic acid amplification cleanup;
(xvii) nucleic acid amplification;
(xviii) post-nucleic acid amplification cleanup;
(xix) pre-nucleic acid sequencing cleanup;
(xx) nucleic acid sequencing;
(xxi) post-nucleic acid sequencing cleanup;
(xxii) reverse transcription;
(xxiii) pre-reverse transcription cleanup;
(xxiv) post-reverse transcription cleanup;
(xxv) nucleic acid ligation;
(xxvi) nucleic acid hybridization;
or
(xxvii) quantification;
and
the separation chamber comprises a detection position;
and
(b) a separation and detection system comprising,
(i) a separation element for simultaneously separating a plurality of target analytes in the separation chamber;
(ii) one or more light sources positioned to illuminate the detection positions on the biochip;
(iii) one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions;
and
(iv) a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components and, the wavelength dispersive element is positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein
each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements;
and wherein said light detector detects fluorescence from at least 6 dyes labeled to one or more biological molecules, each dye having a unique peak wavelength.

25 more elements

An integrated biochip system comprising:

(a) a biochip comprising one or a plurality microfluidic systems, wherein each microfluidic system comprises a first reaction chamber in microfluidic communication with a separation chamber, wherein
the first reaction chamber is adapted for
(i) nucleic acid extraction;
(ii) nucleic acid purification;
(iii) pre-nucleic acid amplification cleanup;
(iv) nucleic acid amplification;
(v) post-nucleic acid amplification cleanup;
(vi) pre-nucleic acid sequencing cleanup;
(vii) nucleic acid sequencing;
(viii) post-nucleic acid sequencing cleanup;
(ix) reverse transcription;
(x) pre-reverse transcription cleanup;
(xi) post-reverse transcription cleanup;
(xii) nucleic acid ligation;
(xiii) nucleic acid hybridization;
or
(xiv) quantification;
and
the separation chamber comprises a detection position;
and
(b) a separation and detection system comprising,
(i) a separation element for simultaneously separating a plurality of biological molecules comprising DNA sequences, in the separation chamber;
(ii) one or more light sources positioned to illuminate the detection positions on the biochip;
(iii) one or a plurality of first optical elements positioned for collecting and directing light emanating from the detection positions;
and
(iv) a light detector positioned to accept light directed from the one or plurality of first optical elements, wherein the light detector comprises a wavelength dispersive element to disperse the light from the one or plurality of first optical elements according to light wavelength into at least 6 wavelength components, and the wavelength dispersive element positioned to provide at least a portion of the dispersed at least 6 wavelength components to at least 6 detection elements, wherein each of the detection elements is in communication with a first control element for simultaneously collecting detection information from each of the detection elements;
and
wherein said light detector detects fluorescence from at least 8 dyes labeled to one or more DNA sequences, each dye having a unique peak wavelength, said dyes being members of at least two 4-dye containing subsets, such that said dye sets are capable of detecting at least two DNA sequences in a single channel, wherein the number of dyes is a multiple of four, and the number of DNA sequences to be detected is equal to one quarter of the multiple, such that each of the different dyes is present in only one subset.

25 more elements

Dependencies are inferred from the claim text.

Integrated Nucleic Acid Analysis

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Integrated Nucleic Acid Analysis

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Abstract

Claims45